Encapsulated cells which produce a biologically active molecule, when implanted in a host, may be used to prevent or treat many diseases or disorders or to provide, restore or augment one or more metabolic functions in the host.
One approach to encapsulating cells is called "microencapsulation", wherein tiny spheres encapsulate a microscopic droplet of a cell-containing solution (Sefton et al., Biotechnology and Bioengineering 29, pp. 1135-1143 (1987); Sugamori et al., Trans. Am. Soc. Artf. Intern. Organs 35, pp. 791-799 (1989)).
Another approach to encapsulating cells, "macroencapsulation" involves encapsulating a plurality of cells in a thermoplastic capsule. Typically this is accomplished by loading cells into a hollow fiber and then sealing the extremities. Various types of macrocapsules are known in the art. In particular, Dionne et. al. (WO 92/19195) refers to a macrocapsule having cells dispersed in a matrix and a semipermeable surface jacket, and is incorporated herein by reference. See also Aebischer, U.S. Pat. Nos. 5,158,881, 5,283,187 and 5,284,761 which refer to a cell capsule formed by co-extruding a polymer solution and a cell suspension.
Typically, when the cells used for encapsulation and implantation are isolated directly from tissue (primary cells), they are disaggregated, washed, and then encapsulated. See, e.g., Aebischer et al., Trans. Am. Soc. Artif. Intern. Organs, 32, pp. 134-7 (1986); Altman et al., Diabetes, 35, pp. 625-33 (1986); Chang et al., U.S. Pat. No. 5,084,350); Darquay and Reach, Diabetologia, 28, pp. 776-80 (1985); Sugamori and Sefton, Trans. Am. Soc.Artif. Intern. Organs, 35, pp. 791-9 (1989).
When immortalized cells or cell lines are to be encapsulated and implanted, they are typically isolated from nutrient-rich cultures. See e.g., Aebischer et al., Biomaterials, 12, pp. 50-55 (1981); Experimental Neurology, 111, pp. 269-75 (1981) (dopamine-secreting PC12 cells), and Ward et al, WO 93/22427 (IgG-secreting MOPC-31C cells).
Encapsulated cells are usually incubated in vitro and functionally characterized before implantation. Encapsulated cells are often cultured in a defined medium during this pre-implantation stage. Often the medium is a balanced salt solution lacking nutrient additives (e.g. Aebisher, supra; Altman, supra; Chang et al., supra). Alternatively, encapsulated cells are incubated in a nutrient medium such as RPMI 1640, which contains various amino acids, vitamins, inorganic salts and glucose (2 g/L; 11.11 mM) (Animal Cell Culture, Eds. Pollard and Walker, Humana Press Inc., Clifton, N.J., pp. 696-700 (1990)), and is typically supplemented with 5%-15% fetal calf or horse serum.
Cells that are encapsulated and implanted in a host must undergo at least two severe changes in nutrient conditions as compared to in vitro conditions. The first occurs upon encapsulation.
Compared to in vitro conditions, cells in an encapsulated environment are nutrient depleted. This depletion is manifested in two ways. There is a nutrient gradient between the external environment and capsule interior which naturally forms across the membrane. This gradient is further accentuated because molecules do not diffuse freely between the outside host tissue and the cells at every position within the capsule. Cells closer to the capsule surface have preferential access to nutrients diffusing across the capsule jacket. In addition, waste products of cells closer to the capsule surface are more readily eliminated.
A second severe change in the concentration of nutrients, e.g., oxygen and glucose, occurs upon implantation in a host. This is because in vitro oxygen and at least some other nutrient levels are generally much higher than occurs in vivo. Thus the driving force for diffusion of these molecules into the capsule is diminished in vivo.
These changes in the nutrient environment may result in an alteration of one or more cell properties. For example, cell death or reduced long term cell viability can result. In addition, the change in environmental conditions upon implantation may also affect other properties of remaining viable encapsulated cells, such as cell growth rate or the cells' ability to produce a biologically active molecule. Changes in the growth rate of discrete subpopulations of cells may result in a takeover of the capsule by a faster growing subpopulation of cells, potentially leading to an apparent shift in the capsule output characteristics, or other potentially undesirable effects.
One important difference between in vivo implantation conditions and in vitro conditions is the glucose concentration to which the cultured cells are adapted. Another difference between conditions in tissue culture and those at an implantation site is the amount of oxygen available to the cells. Other nutrients may be at significantly different concentrations in culture medium and at a given implantation site.
Cells or tissues that are highly active metabolically are particularly susceptible to the effects of nutrient and oxygen deprivation (hypoxia). Likewise, many endocrine tissues that are normally sustained by dense capillary beds and are thus acclimated to growth in high oxygen and nutrient levels in vivo exhibit this behavior; pancreatic islets of Langerhans and adrenal chromaffin cells are particularly sensitive to hypoxic shock.
Changes in oxygen tension and nutrient stress are known to alter the expression of a large number of genes that affect a variety of cellular functions. Such changes can affect the stability and function of certain mRNAs. For example, the tyrosine hydroxylase mRNA, which encodes the enzyme that catalyzes the rate limiting step in dopamine production, may be affected by nutritional stress.
Further, various heat shock genes, and the expression of metabolic enzymes like those involved in intermediary metabolism (e.g. the glycolytic and gluconeogenic pathways) may be affected by low glucose or amino acid levels.
Importantly, highly-differentiated cell types that are deprived of oxygen can lose their tissue-specific functions until they recover from hypoxic shock, [see, e.g., Wolffe and Tata, FEBS Letters, 176, pp. 8-15 (1984)]. Functions that are lost or diminished include the synthesis and modification of proteins. This may affect the production and secretion of the very therapeutic factors that cells are intended to supply to the surrounding host tissue. In addition, hypoxic conditions can in some cases initiate malignant transformation, (see, e.g., H. Goldblatt et at., Biochemical Medicine, 7, pp. 241-52 (1973)).
It is desirable to develop a method for implantation involving exposing or acclimating cells to one or more restrictive conditions prior to implantation to reduce alterations in cell properties resulting from the effect of the change in environmental conditions upon implantation, and thus reduce any adverse consequences to the host. It is also desirable to develop cell lines of the cells so prepared. It is also desirable to provide a means for ex vivo study of cells which have undergone changes in the in vivo environment.